ß-carotene-producing bacteria residing in the intestine provide vitamin A to mouse tissues in vivo.

Vitamin A deficiency (VAD) is an overwhelming public health problem that affects hundreds of millions of people worldwide. A definitive solution to VAD has yet to be identified. Because it is an essential nutrient, vitamin A or its carotenoid precursor ß-carotene can only be obtained from food or supplements. In this study, we wanted to establish whether ß-carotene produced in the mouse intestine by bacteria synthesizing the provitamin A carotenoid could be delivered to various tissues within the body. To achieve this, we took advantage of the Escherichia coli MG1655*, an intestine-adapted spontaneous mutant of E. coli MG1655, and the plasmid pAC-BETA, containing the genes coding for the 4 key enzymes of the ß-carotene biosynthetic pathway (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase) from Erwinia herbicola. We engineered the E. coli MG1655* to produce ß-carotene during transformation with pAC-BETA (MG1655*-ßC) and gavaged wild-type and knockout mice for the enzyme ß-carotene 15,15'-oxygenase with this recombinant strain. Various regimens of bacteria administration were tested (single vs. multiple and low vs. high doses). ß-Carotene concentration was measured by HPLC in mouse serum, liver, intestine, and feces. Enumeration of MG1655*-ßC cells in the feces was performed to assess efficiency of intestinal colonization. We demonstrated in vivo that probiotic bacteria could be used to deliver vitamin A to the tissues of a mammalian host. These results have the potential to pave the road for future investigations aimed at identifying alternative, novel approaches to treat VAD.

Processive pectin methylesterases: the role of electrostatic potential, breathing motions and bond cleavage in the rectification of Brownian motions.

Pectin methylesterases (PMEs) hydrolyze the methylester groups that are found on the homogalacturonan (HG) chains of pectic polysaccharides in the plant cell wall. Plant and bacterial PMEs are especially interesting as the resulting de-methylesterified (carboxylated) sugar residues are found to be arranged contiguously, indicating a so-called processive nature of these enzymes. Here we report the results of continuum electrostatics calculations performed along the molecular dynamics trajectory of a PME-HG-decasaccharide complex. In particular it was observed that, when the methylester groups of the decasaccharide were arranged in order to mimic the just-formed carboxylate product of de-methylesterification, a net unidirectional sliding of the model decasaccharide was subsequently observed along the enzyme's binding groove. The changes that occurred in the electrostatic binding energy and protein dynamics during this translocation provide insights into the mechanism by which the enzyme rectifies Brownian motions to achieve processivity. The free energy that drives these molecular motors is thus demonstrated to be incorporated endogenously in the methylesterified groups of the HG chains and is not supplied exogenously.

Cold active pectinases: advancing the food industry to the next generation.

Pectinase has been an integral part of commercial food processing, where it is used for degradation of pectin and facilitates different processing steps such as liquefaction, clarification and juice extraction. The industry currently uses pectinases from mesophilic or thermophilic microorganisms which are well established, but recently, there has been is a new trend in the food industry to adopt low-temperature processing. This trend is due to the potential economic and environmental advantages which the industry envisages. In order to achieve this change, an alternative for the existing pectinases, which are mostly mesophilic and temperature-dependent, must be identified, which can function efficiently at low temperatures. Psychrophilic pectinases derived from cold-adapted microorganisms, are known to function at low to freezing temperatures and may be an alternative to address the problem. Psychrophilic pectinases can be obtained from the vast microflora inhabiting various cold regions on earth such as oceans, Polar Regions, snow-covered mountains, and glaciers. This article is intended to study the advantages of cold active pectinases, its sources, and the current state of the research.

Substrate dynamics in enzyme action: rotations of monosaccharide subunits in the binding groove are essential for pectin methylesterase processivity.

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme's binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.

Immobilization of glucosyltransferase from Erwinia sp. using two different techniques.

Two different techniques of glucosyltransferase immobilization were studied for the conversion of sucrose into isomaltulose. The optimum conditions for immobilization of Erwinia sp. glucosyltransferase onto Celite 545, determined using response surface methodology, was pH 4.0 and 170 U of glucosyltransferase/g of Celite 545. Using this conditions more than 60% conversion of sucrose into isomaltulose can be obtained. The immobilization of glucosyltransferase was also studied by its entrapment in microcapsules of low-methoxyl pectin and fat (butter and oleic acid). The non-lyophilized microcapsules of pectin, containing the enzyme and fat, showed higher glucosyltransferase activity, compared with lyophilized microcapsules containing enzyme plus fat, and also lyophilized microcapsules containing enzyme without fat addition. The non-lyophilized microcapsules of pectin containing the glucosyltransferase and fat, converted 30% of sucrose into isomaltulose in the first batch. However the conversion decreased to 5% at the 10th batch, indicating inactivation of the enzyme.

Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.

Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

Cloning and characterization of the gene cluster for palatinose metabolism from the phytopathogenic bacterium Erwinia rhapontici.

Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class III. Using immunoblotting experiments, we detected PelI homologs in various strains of E. chrysanthemi and E. carotovora subsp. carotovora but not in E. carotovora subsp. atroseptica.

A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases.

A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene. The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening.

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates.

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229-1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and 13C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyro-phosphorylase antisense plants was observed.

Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

Expression of the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains capable of simultaneous secretion of bacterial glucanase and pectinase enzymes have been developed. The Butyrivibrio fibrrisolvens endo-beta-1,4-glucanase gene (end1), the Erwinia chrysanthemi pectate lyase gene (pelE) and E. carotovora polygalacturonase gene (peh1) were each inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into yeast-centromeric shuttle vectors. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of glucanase and pectinases was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). These YCplac111-based constructs, designated END1, PEL5, AND PEH1, respectively, were transformed into S. cerevisiae. The END1, PEL5 and PEH1 constructs were co-expressed in laboratory strains of S. cerevisiae as well as in wine and distillers' yeasts. DNA-RNA hybridization analysis showed the presence of END1, PEL5 and PEH1 transcripts. Carboxymethylcellulose and polypectate agarose assays revealed the production of biologically active endo-beta-1,4-glucanase, pectate lyase and polygalacturonase by the S. cerevisiae transformants. Interestingly, although the same expression-secretion cassette was used in all three constructs, time-course assays indicated that the pectinases were secreted before the glucanase. It is tempting to speculate that the bulkiness of the END1-encoded protein and the five alternating repeats of Pro-Asp-Pro-Thr(Gln)-Pro-Val-Asp within the glucanase moiety could be involved in the delayed secretion of the glucanase.

Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.

The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005.

Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16.

A 14-kilobase BamHI-EcoRI DNA fragment cloned from Erwinia chrysanthemi EC16 contained a gene encoding a metalloprotease inhibitor as well as three tandem prt genes encoding metalloproteases. The prt genes were separated from the inhibitor gene by a ca. 4-kilobase region that was necessary for extracellular secretion of the proteases. When individually subcloned downstream from vector promoters, the three prt genes each led to substantial extracellular secretion of the proteases by Escherichia coli cells, provided that the 4-kilobase required region was supplied in cis or trans. One of the protease structural genes, prtC, was sequenced and had high homology to a metalloprotease gene previously described from Serratia species as well as to the prtB gene of E. chrysanthemi B374. Marker exchange mutants of E. chrysanthemi EC16 defective in production of one or all of the extracellular proteases were not impaired in virulence on plant tissue.

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia.

A set of cDNAs that are expressed in tomato anthers were isolated. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica.

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of homology between the pectate lyase-encoding pelB and pelC genes in Erwinia chrysanthemi.

The genes for two of several pectate lyase isozymes produced by the phytopathogenic enterobacterium Erwinia chrysanthemi 1237 were subcloned and compared by DNA-DNA hybridization, and the encoded proteins were analyzed. The borders of the genes were located on a restriction map by incremental exonuclease III deletions. DNA-DNA hybridization studies revealed a low percentage of mismatch (7 to 17%) between pelB and pelC. No homology was detected between pelC and other regions of the E. chrysanthemi 1237 chromosome, in which three other isozyme genes apparently reside. The pectate lyase isozymes were readily purified by chromatofocusing or granulated-gel bed isoelectric focusing from the periplasmic shock fluids of Escherichia coli subclones. The molecular weights of PLb and PLc were 30,000 and 33,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their isoelectric points were 7.6 and 8.1, respectively, as determined by equilibrium isoelectric focusing in ultrathin polyacrylamide gels. The Km values for PLb and PLc were 0.20 and 0.32 mg/ml, respectively, with polygalacturonate as a substrate. Thin-layer chromatography of reaction products and viscometric assays revealed little difference between the two isozymes. All our data indicate that the genes are duplicates and that the proteins are isofunctional.

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.